Sample pretreatment is often a desired preliminary step prior to analysis of complex samples of biological origin. Analytes can be present at insufficient concentration, precluding detection or requiring application of excessive sample volumes to the analytical instrument. For example, in separation methods such as gas chromatography (GC), capillary electrophoresis (CE), capillary electrochromatography (CEC) and high performance liquid chromatography (HPLC), sample volumes are in the range of nL to μL, requiring that analytes be present in amounts sufficient for detection in those volumes. In addition, contaminants such as salts, metals, buffers, proteins, etc. can be present in complex samples and could interfere with or damage sensitive instruments. Therefore, pretreatment of samples is usually necessary prior to analysis of the samples.
Sample preparation is the most time consuming step in the analysis of drugs or other active agents present in the concentration range of pg/mL to μg/mL in biological samples such as serum, urine, blood, water samples, etc. Sample preparation procedures are needed that are fast and simple to perform, provide good recovery of analytes from interfering contaminants, and concentrate analytes so that sufficient quantities of analytes are present in the desired volume for analysis. Ideally, the sample preparation step is able to trap analytes in 1-50 μL of solvent that can be directly injected into the analytical instrument. For example, for GC, it is preferred that the analytes are concentrated into an organic solvent that can be introduced into the gas chromatograph, while for CE or HPLC, aqueous solvents are preferred.
The most frequently used extraction techniques are liquid-liquid extraction (LLE) and solid-phase extraction (SPE), resulting in concentration of analytes into solvent volumes of 0.2 to 10 mL of extraction solvent based on partitioning of analytes into the acceptor phase. However, for small sample volumes, these techniques result in poor recovery of analytes and/or analyte concentrations that are too low for analysis. As discussed in WO 00/33050, quantitative extraction using LLE can only be achieved by using large volumes of extraction solvent relative to the sample volume. Generally a volume of solvent of from one half to ten times the volume of the sample is used, which results in a need for an additional concentration step, and further increases the time involved in sample preparation and reduces analyte recovery. Using SPE, 100 mg of adsorbent is generally required to extract analytes from a 1 mL sample, and results in a maximum concentration enhancement of a factor of 4, assuming quantitative recovery from the solid phase adsorbent. It is desired to purify and concentrate analytes in a solvent by a factor of 10 to 100 or more, which is not possible using LLE or SPE.
Microextraction procedures provide one partial solution to these problems. In microextraction procedures, the analytes are extracted from a large volume of sample into a smaller volume of an acceptor phase. The acceptor phase can be a solid phase, as in solid-phase microextraction (SPME), an organic solvent as in liquid-liquid microextraction (LLME) or an aqueous solvent as in liquid-liquid-liquid microextraction (LLLME) also known as liquid phase microextraction (LPME), wherein uncharged analytes are first equilibrated with an intermediate organic phase, then trapped in an aqueous acceptor phase in a charged and impermeable form which cannot traverse across the organic phase back to the sample solution.
Adsorption onto the solid phase in SPME is accomplished using a solid polymer coated onto a fiber. The polymer acceptor phase is nonvolatile and has a volume less than 1 μL. However, when applied to trace analysis of organic compounds from complex biological samples, enrichment from the biological matrix is reduced relative to enrichment from a water sample, due to the reduced capacity of the acceptor phase.
Liquid phase microextraction (LPME) encompasses both LLME and LLLME, and overcomes many of the limitations of solid phase extraction and microextraction. For example, as shown in FIG. 1 of WO 00/33050, extraction of an analyte from a sample containing 1 μg/mL of the analyte results in a concentration at equilibrium of 0.99 μg/mL in the acceptor phase using LLE and a concentration of 50 μg/mL in the acceptor phase using LLME, when the partition coefficient between the aqueous phase and the acceptor phase is 100. As demonstrated in Table 1 of WO 00/33050, enrichment of analyte in the acceptor phase depends on the volume of the acceptor phase and the partition coefficient. Enrichments of from about 1 to 500 are possible from a sample containing 1 μg/mL of analyte into acceptor phases with partition coefficients of 10 to 1000, respectively.
It is desirable to keep the acceptor phase separate from the sample in order to facilitate recovery of the acceptor solution and the enriched analytes. The acceptor phase can be maintained separate from the donor or sample phase by, for example, incorporating the acceptor phase into sponges, from which it can be removed after extraction is complete, by filling a hollow fiber of porous polymer with the acceptor phase, from which it can be withdrawn after extraction is complete, or by forming a liquid membrane on the surface of a hollow fiber of porous polymer where the ultimate acceptor phase is aqueous and is present on the interior of the hollow fiber. The aqueous acceptor phase enriched in analytes can then be removed after extraction is complete. The first two approaches (LLME) allow the enrichment of the analyte directly into the acceptor phase. The latter approach (LLLME) also allows the selective enrichment of acidic and basic compounds. For example, enrichment of a carboxylic acid analyte is effected from a sample by adjusting the pH of the sample solution below the pKa of the acid so that the carboxylic acid is neutralized and partitions into the liquid membrane, while the pH of the acceptor aqueous solution is above the pKa of the acid, causing the analyte diffusing into the acceptor solution to be trapped in its charged form. The principles of LLLME and LLME are discussed in detail in WO 00/33050.
WO 02/088672 discloses supported liquid membranes, for example, porous membrane supports, which are impregnated with a water insoluble organic solvent, for performing LLLME. Porous hollow fiber or porous-disk liquid membrane devices and methods of use are described that allow purification and enrichment by factors of several hundred for analytes of interest from biological fluid samples. However, for this technique to work optimally, stable membranes are required which are also permeable to analytes of varying hydrophobicity. The devices and methods described in WO 02/088672 are reported to utilize solvents that are immiscible with water as liquid membranes, including aliphatic or aromatic hydrocarbons, ethers, nitrites, aldehydes or ketones, and alcohols. This reference reports that the most stable membranes were formed when using hydrophobic liquids, such as pure hydrocarbons (e.g., dodecane), while the greatest diffusion and hence optimal analyte enrichment occurred when utilizing more polar solvents. A balance between stability and analyte diffusion was sought by mixing the solvents to achieve stable liquid membranes with high diffusion coefficients to analytes. However, WO 02/088672 also discloses that, once prepared, these supported liquid membranes generally have limited lifetimes. One solvent, nitrophenyloctylether, was reported to provide a membrane lifetime of 10-20 days, while the others tested possessed lifetimes of at most 5 days.
Therefore, there is a need in the art for more stable and permeable liquid membrane devices for performing LPME. There is also a need in the art for improved methods of preparing and using these devices in the performance of LPME.